The present invention relates to a viral vector comprising a nucleic acid encoding pigment epithelium derived factor.
Gene therapy is the next best hope for the treatment and prevention of a broad array of human diseases. Since the first gene therapy clinical trials started in 1990, more than 300 clinical protocols have been approved and an estimated $200 million is spent by the National Institutes of Health (NIH) to develop the tools and techniques necessary to practice gene therapy (Marshall, Science, 269, 1050-1055 (1995); and Anderson, Nature, 392, 25-30 (1998)). The potential of treating disease at its most basic level is staggering. Yet, many hurdles exist in genetic research before the unlimited potential of gene therapy can be realized in the clinic. Obstacles associated with the wide-spread acceptance of gene therapy include obtainment of long term gene expression, efficient nucleic acid delivery, transduction of both dividing and non-dividing cells, target cell specificity, safety, and construction of inexpensive expression vectors for use in gene therapy protocols. Gene therapy research can be divided into two major areas: techniques and tools. Gene therapy techniques that are currently under study include methods of gene transfer in vivo and ex vivo, cell culture methods, methods of identifying appropriate target cells, methods of quantifying and qualifying gene expression in vivo, and the like.
In addition to perfecting techniques employed in gene therapy, much research has been focused on the tools of gene transfer for the purpose of treating or preventing disease. Many in the art have called for additional research into the improvement of gene transfer vectors, regulatory sequences, and producer cell lines. A great deal of enthusiasm surrounds the use of synthetic gene delivery vehicles and naked DNA. In fact, the first expression vector used for gene transfer was naked DNA. Naked DNA, e.g., plasmids, have virtually unlimited capacity and are relatively simple to construct. Plasmids are genetically engineered circular double-stranded DNA molecules that are inexpensive and easy to produce, and can transduce any type of gene or functional nucleic acid into cells. Yet, the level of expression efficiency of plasmids is poor, plasmids are not easily taken up by host cells, and plasmids are easily degraded when exposed to high temperatures, enzymes, chemicals, mechanical stress, and the like. Thus, to increase the efficiency of gene transfer and vector stability, naked DNA is often complexed with liposomes or other molecules. Liposomes are vesicle-type structures wherein fluid is encapsulated by a lipid bilayer. While the liposomes used for plasmid-mediated gene transfer strategies have various compositions, they are typically synthetic cationic lipids. Due to the negative charge of DNA, naked DNA is attracted to the positively-charged surface of liposomes. Naked DNA also can be conjugated to other molecules, such as proteins, in order to facilitate DNA uptake. The proteins associated with DNA allow targeting of the nucleic acid molecule to a particular cell type, as well as increase plasmid uptake. Liposome- and molecular conjugate-mediated gene transfer is a great deal more efficient than transfection of non-complexed, naked DNA.
Clearly, several advantages exist in using naked DNA in gene therapy protocols. Plasmids are largely undetected by the body""s innate immune system and, therefore, are not readily cleared by the body. In addition, plasmids are non-infectious and are rarely mutagenic. As such, naked DNA is believed by some to be the ideal mode of gene transfer for purposes of gene therapy. Yet, even when complexed with facilitators, efficiency of host cell transfection and subsequent expression of transgenes is relatively low. For instance, although liposome-mediated gene transfer may introduce plasmids into host cells, the majority of the transferred DNA is lost, most likely due to lysosomal degradation (French, Herz, 18, 222-229 (1993)). As sufficient expression of therapeutic genes to treat disease is a major obstacle in gene therapy, other means of gene transfer are needed in order to ensure the success of gene therapy protocols.
In addition to the identification and development of ideal vectors, another tool needed for the success of gene therapy in the clinic is therapeutic factors and the nucleic acids that encode them. Ideally, a therapeutic factor for use in gene therapy has utility in treating a number of afflictions, and can be delivered and expressed in vivo. One such factor, pigment epithelium-derived factor (PEDF), has recently been identified and realized to have both neurotrophic and anti-angiogenic properties. Regrettably, PEDF is almost solely generated in human fetus retinal cells. The poor production of PEDF from retinal pigment epithelial (RPE) cells and the scarcity of the source tissue of PEDF complicates the use of this potentially valuable therapeutic factor in the clinic.
Given the hurdles associated with gene therapy, in particular the difficulties associated with efficient expression of appropriate therapeutic factors, there remains a need in the art for an expression vector comprising a coding sequence for a therapeutic factor that potentially can aid in the treatment of a number of afflictions. The present invention provides such an expression vector. This and other advantages of the present invention will become apparent from the detailed description provided herein.
The present invention provides a viral vector comprising a nucleic acid sequence encoding pigment epithelium-derived factor (PEDF) or a therapeutic fragment thereof. The nucleic acid sequence is operably linked to regulatory sequences necessary for expression of PEDF or a therapeutic fragment thereof. Preferably, the viral vector is an adenoviral vector or an adeno-associated viral vector. Also preferably, the viral vector further comprises one or more additional nucleic acid sequences encoding therapeutic substances other than PEDF or a therapeutic fragment thereof.